Molecular cloning and characterization of TK1111, a cupin-type phosphoglucose isomerase from Thermococcus kodakarensis

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1 DERNEĞİ BİYOKİMYA DERGİSİ TÜRK TÜRK BİYOKİMYA DERNEĞİ DERGİSİ TÜRK BİYOKİMYA DERNEĞİ DERGİSİ ORJİNAL Türk Biyokimya Dergisi [Turkish Journal of Biochemistry Turk J Biochem] 2013; 38 (4) ; doi: /tjb Research Article [Araştırma Makalesi] Yayın tarihi 30 Aralık, 2013 TurkJBiochem.com [Published online 30 December, 2013] Molecular cloning and characterization of TK1111, a cupin-type phosphoglucose isomerase from Thermococcus kodakarensis [Bir Cupin-tip fosfoglukoz izomeraz olan TK1111 in Thermococcus kodakarensis den moleküler klonlanması ve karakterizasyonu]* ÖRNEK 2. ÖRNEK 1976 Shahzada Nadeem Abbas 1, Naeem Rashid 1, Iram Aziz 1, Muhammad Akhtar 1,2 1 School of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan; 2 School of Biological Sciences, University of Southampton, Southampton SO16 7PX, England ABSTRACT Aim: To characterize, TK1111, a cupin-type phosphoglucose isomerase from a hyperthermophilic archaeon Thermococcus kodakarensis. Methods: The genome of T. kodakarensis was searched in order to find an open reading frame annotated as glucose-6-phosphate isomerase. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Effect of metal ions on the restoration of enzyme activity was examined. Results: Genome search of T. kodakarensis revealed the presence of an open reading frame, TK1111, annotated as glucose-6-phosphate isomerase. TK1111 exhibited high homology with archaeal PGIs related to the cupin-superfamily instead of well-known family of PGI enzymes. The gene encoding TK1111 consisted of 567 nucleotides corresponding to a polypeptide of 189 amino acids. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Recombinant TK1111 displayed highest activity (43 U/mg) at 90 C and ph 6.0. To our knowledge this is the only PGI from cupin-superfamily exhibiting highest activity in the acidic ph. TK1111 catalyzed the conversion of fructose 6-phosphate to glucose 6-phosphate following Michaelis-Menten kinetics with a K m and V max values of 2.2 mm and 12.5 mmol min -1 mg -1, respectively at 37 C. Addition of Zn 2+ ions in the assay mixture remarkably enhanced the enzyme activity in contrast to the PGI from Thermococcus litoralis, another member of the same genus exhibiting 75% identity to TK1111. The enzyme activity was completely diminished when TK1111 was heated at 70 C in the presence of EDTA. Complete restoration of the enzyme activity was observed when TK1111 was incubated at room temperature in the presence of Zn 2+. Conclusion: TK1111 from T. kodakaraensis is a novel phosphoglucose isomerase that belongs to cupintype isomerases. To our knowledge this is the only PGI from cupin-family exhibiting highest activity in the acidic ph. Enzyme activity was remarkably enhanced in the presence of Zn 2+ in contrast to other members of the family. Key Words: Phosphoglucose isomerase, metalloenzyme, cupin-family, thermostability, Thermococcus kodakarensis, hyperthermophile, archaeon Conflict of Interest: There is no conflict of interest between the authors. Yazışma Adresi [Correspondence Address] Naeem Rashid School of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan Tel Fax *Translated by [Çeviri] Dr. Samiye Yabanoğlu Registered: 10 September 2013; Accepted: 7 November 2013 [Kayıt Tarihi: 10 Eylül 2013; Kabul Tarihi: 7 Kasım 2013] ÖZET Amaç: Hipertermofilik bir archeon olan Thermococcus kodakarensis den, bir cupin-tip fosfoglukoz izomeraz olan TK1111 in karakterizasyounun yapılması. Yöntemler: T. kodakarensis in genomu glukoz-6-fosfat izomeraz için bir açık çerçeve bulmak amacıyla araştırılmıştır. Yapısal gen klonlanarak, Escherichia coli de eksprese edilmiş ve saflaştırılan gen ürünü karakterize edilmiştir. Metal iyonlarının enzim aktivitesinin yenilenmesi üzerine etkileri incelenmiştir. Bulgular: T. kodakarensis in genom araştırması, glukoz-6-fosfat izomeraz için tanımlanan bir açık çerçeve, TK1111, varlığını ortaya çıkartmıştır. TK1111, iyi bilinen PGI enzim aileleri yerine, cupin-süperailesiyle ilişkili olan archeal PGIlarla yüksek homoloji göstermiştir. TK1111 ü kodlayan gen, 189 amino asitlik bir polipeptidle ilişkili 567 nükleotid içermektedir. Yapısal gen klonlanarak, Escherichia coli de eksprese edilmiş ve saflaştırılan gen ürünü karakterize edilmiştir. Rekombinant TK1111 en yüksek aktiviteyi (43 U/mg) 90 C ve ph 6.0 da göstermiştir. Bilgilerimize dayanarak cupin-süperailesindeki PGI lar arasında, en yüksek aktivitesini asidik ph da gösteren tek enzimdir. TK1111, Michealis-Menten kinetiğini takip ederek, fruktoz-6-fosfatın glukoz-6-fosfata çevrimini katalizler ve 37 C de sırasıyla 2.2 mm ve 12.5 mmol min -1 mg -1 K m ve V max değerlerine sahiptir. Reaksiyon ortamına Zn 2+ iyonlarının eklenmesi, aynı genusun başka bir üyesi olan ve TK1111 e %75 benzerlik gösteren, Thermococcus litoralis den elde edilen PGI nın aksine, enzim aktivitesini belirgin biçimde arttırmıştır. TK1111, EDTA varlığında 70 C ye ısıtıldığında enzim aktivitesi tamamen azalmıştır. TK1111 oda sıcaklığında ve Zn 2+ va rl ığ ı nd a inkübe ediliğinde enzim aktivitesinin tamamen geri kazanılmıştır. Sonuç: T. kodakaraensis den elde edilen TK1111, cupin-tip izomerazlara dahil olan yeni bir fosfoglukoz izomerazdır. Bilgilerimize göre cupin-ailesi içinde, en yüksek aktivitesini asidik ph da gösteren tek PGI dır. Ailenin diğer üyelerinin aksine, enzim aktivitesi Zn 2+ varlığında belirgin biçimde artış göstermiştir. Anahtar Kelimeler: Fosfoglukoz izomeraz, metaloenzim, cupin-ailesi, termostabilite, Thermococcus kodakarensis, hipertermofil, archaeon Çıkar Çatışması: Yazarların çıkar çatışması bulunmamaktadır ISSN X (electronic) (printed)

2 Introduction Phosphoglucose isomerase (PGI; EC ) catalyses the reversible isomerization of glucose-6-phosphate (G6P), an aldose, and fructose-6-phosphate (F6P), a ketose. PGI is an important player of sugar metabolism in living organisms. Majority of the PGIs belong to the PGI superfamily, consisting of the PGI family and the bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI) family [1]. PGIs belonging to the PGI superfamily are found in all the three domains of life and are extensively studied. Bifunctional PGIs/ PMIs are mainly found in the crenarchaeotal branch of archaea [2]. A third and a novel type of PGI has been found in euryarchaeotal branch of archaea [1, 3, 4] and it belongs to the cupin-family comprising a group of functionally diverse proteins that contain a central domain forming a small barrel called cupin. Most of the cupin enzymes are metallo enzymes having two conserved sequences(consensus, G(X) 5-8 H(X)H(X) E(X) G, motif I; and G(X) P(X) H(X) 3- N, motif II)The PGIs belonging to this group have been characterized from archaea including Archaeoglobus fulgidus [5], Methanosarcina mazei [5], Pyrococcus furiosus [1, 3], and Thermococcus litoralis [4] as well as from bacteria including Salmonella enterica and Ensiferm eliloti [5]. Determination of active-site residues of PGI from P. furiosus has been accomplished by docking of monosaccharides onto the structure models of the enzyme [6]. Thermococcus kodakarensis is a hyperthermophilic euryarchaeon isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan [7, 8]. The strain is a strict anaerobe and it can grow heterotrophically on starch, pyruvate, amino acids and peptides. Whole genome of T. kodakarensis has been sequenced and reported [9]. The genome sequence revealed the presence of an open reading frame exhibiting high homology to the cupin-type PGIs. We describe here cloning of the PGI gene (TK1111) from T. kodakarensis, and characterization of the gene product produced in Escherichia coli. Materials and Methods Materials Restriction enzymes, DNA and protein markers, TA cloning and DNA extraction kits, Taq DNA polymerase (recombinant), and ptz57r/t cloning vector were purchased from Thermo Scintific (Thermo Scintific, MA, USA). Expression vectors pet-21a(+) was obtained from Stratagene (Stratagene, La Jolla, CA, USA), E. coli DH5α and BL21 Codon Plus cells, were purchased from Novagen (Novagen, Madison, WI, USA). Primers were synthesized from e-oligos (Gene Link, NY, USA). All other chemicals were purchased from Sigma (Sigma- Aldrich, Taufkirchen, Germany). Homology comparison and phylogenetic tree construction Database homology search was performed by Basic Local Alignment Search Tool (BLAST) program [10]. Multiple sequence alignment of TK1111 homologs was performed and a neighbor-joining tree of the aligned sequences was generated with ClustalW [11] provided at: (http://clustalw.ddbj.nig.ac.jp/). Cloning of TK1111 The structural gene, TK1111, encoding a homolog of cupin-type PGI, was amplified by polymerase chain reaction (PCR) using genomic DNA of T. kodakarensis as a template and a sequence specific set of forward (5-GGTGATCATATGGAGTACAAGCGCCC) and reverse (5-GGATTTCGGGCTACTCCTTCCAGC) primers. NdeI restriction enzyme site was introduced in the forward primer (underlined sequence). PCR amplified TK1111 gene was inserted in cloning vector ptz57r/t using T 4 DNA ligase and the resulting plasmid was named as ptz-tk1111. TK-1111 gene was cut from ptz-tk1111 using NdeI (introduced in the forward primer) and HindIII (present in the multicloning site of ptz57r/t) restriction enzymes and ligated in pet-21a at the corresponding sites. The resulting plasmid, pet-tk1111, was used to transform E. coli BL21-CodonPlus(DE3)-RIL for production of TK1111 enzyme. Production in E. coli and purification of recombinant TK1111 E. coli cells harbouring pet-tk1111 were grown to early log phase and expression of the gene was induced with 0.2 mm isopropyl-thio-β-d-galactoside (IPTG). The cells were harvested by centrifugation at 7,000 g for 5 min at 4 C, resuspended in 50 mm Tris-HCl (ph 8.0), and disrupted by sonication at 4 C. The supernatant after centrifugation was heated at 85 C for 25 min. The heat labile proteins from the host cells were denatured and removed by centrifugation at 15,000 g for 20 min at 4 C. The heat-stable recombinant TK1111 in the supernatant was further purified using ÄKTA Purifier chromatography system (GE Healthcare, Uppsala, Sweden). The heat-treated cell extract was applied to anion-exchange Resource Q (6 ml) column (GE Healthcare) and recombinant TK1111 was eluted with a linear gradient of 0 to 1 M NaCl. Analysis of the purified TK1111 was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Measurement of protein concentration The concentration of proteins in solution was determined by Bradford method [12] with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA, USA) using bovine serum albumin as a standard. The standard curve was plotted using 2 to 20 μg of bovine serum albumin in 1 ml of Bio-Rad protein assay reagent. The incubations 439

3 were performed at room temperature for 5 min and the absorbance was measured at 595 nm. Gel electrophoresis DNA samples were electrophoresed on 0.8% agarose gel followed by ethidium bromide staining and visualized under the UV light. Protein samples were analyzed by polyacrylamide gel electrophoresis (PAGE) containing 0.1% sodium dodecyl sulphate (SDS) followed by staining with Coomassie brilliant blue (CBB). TK1111 enzyme activity assay The formation of glucose 6-phosphate from fructose 6-phosphate was determined by measuring the formation of NADPH at 340 nm. The assay mixture contained 50 mm sodium phosphate ph 7.0, 10 mm MgCl 2, 0.5 mm NADP, 3 mm fructose 6-phosphate, and 1.25 units of D-glucose-6-phosphate dehydrogenase. One unit of activity was defined as the amount of enzyme required to convert 1 µmol of fructose 6-phosphate to glucose 6-phosphate per min. The routine enzyme assays were performed at 50 C unless mentioned otherwise. At this temperature the coupling enzymes remained active, and TK1111 was sufficiently active to measure its enzyme activity. The coupling enzymes were present in excess, to ensure that the detected NADPH absorbance at 340 nm ( = 6300 M -1 cm -1 ) corresponds to the PGI activity of TK1111. Effect of temperature and ph on TK1111 activity The temperature optimum was determined in the direction of glucose 6-phosphate formation. Purified recombinant TK1111 was incubated in an assay mixture containing 50 mm sodium phosphate buffer, ph 7.0. The assay mixture was incubated in a water bath at temperatures ranging from 30 to 100 C, pre-heated for 2 min, and the enzyme reaction was started by adding 3 mm fructose 6-phosphate. After 2 min of incubation the reaction was stopped by transferring the assay mixture on ice/ ethanol, and the amount of glucose 6-phosphate formed was determined at room temperature by measuring the reduction of NADP (340 nm) with glucose-6-phosphate dehydrogenase. Corrections were made for the chemical isomerization of fructose 6-phosphate in the absence of TK1111. Optimum ph for TK1111 enzyme activity was determined at 50 C in 50 mm buffers over the ph range Except for buffer and temperature, assay conditions were identical to analyze the temperature optimum. Kinetic parameters Kinetic parameters were determined at 37 C and ph 6.0 with standard assay mixtures containing various concentrations of fructose-6-phosphate. For determination of the K m and V max values, the concentration of the substrate ranged from 0.05 to 5 mm. Results and Discussion Cloning TK1111 gene Polymerase chain reaction for the amplification of TK1111 gene, using the set of primers given in the materials and methods section as priming strands and genomic DNA of T. kodakarensis as template, resulted in the amplification of 0.6 kb DNA fragment (data not shown) exactly matching the size of TK1111 gene. The amplified gene fragment was inserted in ptz57r/t. When the resulting ligation mixture was used to transform E. coli DH5α competent cells, a total of 17 white and 6 blue colonies appeared on the selection plates. White colonies were further screened for the presence of TK1111 gene by polymerase chain reaction (data not shown). Plasmid DNA was isolated from one of the positive clones and digested with NdeI and HindIII which resulted in the liberation of a 0.6 kb DNA fragment from the vector (data not shown). Nucleotide sequence of the insert was determined and the results of DNA sequencing confirmed that the cloned fragment encodes TK1111. Comparison of TK1111 sequence TK1111 gene consisted of 567 nucleotides encoding a protein of 189 amino acids with a calculated molecular mass of 24,675 Da and a pi of 5.1. Amino acid sequence comparison demonstrated that TK1111 exhibits a high degree of similarity to PGIs of the cupin-family. Most cupin-family enzymes are metalloenzymes with two conserved sequences (consensus, G(X)5-8H(X)H(X)3-6E(X)6G, motif I; and G(X)5P(X)4H(X)3N, motif II). These motifs were conserved in TK1111. When we constructed a phylogenetic tree based on the amino acid sequences of various PGIs, TK1111 clustered with PGIs from family Thermococcaceae of archaeal kingdom Euryarchaeota (Fig. 1). Eight active site residues of the cupin-family PGIs (T 63, G 79, H 80, H 82, E 93, Y 95, H 136 and Y 160 ; A. fulgidus numbering) [13] were completely conserved in TK1111. Amino acid sequence comparison showed that TK1111 displayed highest homology of 92% (identity) with uncharacterized PGI originating from Thermococcus zilligii (accession no. WP_ ). Among the characterized PGIs, TK1111 displayed highest homology of 82% with PGI from P. furiosus (accession no. NP_577925). Interestingly, TK1111 displayed relatively lower homology (75%) to PGI characterized from T. litoralis (accession no. WP_ ), a member of the same genus as T. kodakarensis from which TK1111 was originated [4]. Amino acids involved in metal ion binding (H 88, H 90, E 97, and H 136 ; P. furiosus numbering) were completely conserved in TK1111. Similarly, amino acids involved in the formation of two small networks, a 3-residue (K 80, E 175, K 180 ) and a 4-residue (D 94, R 95, D 155, K 92 ) network, for ion pair formation in P. furiosus PGI [14] were completely conserved in TK1111. The 3-residue network 440

4 is missing in T. litoralis PGI as K 80 and K 180 are replaced by N 81 and V 181 (T. litoralis numbering), respectively (Fig. 2). Glutamate at position 45 (E 45, P. furiosus numbering), present in PGIs from T. litoralis and P. furiosus, is missing in TK1111. Apart from the amino acids common in the characterized PGIs from family Thermococcaceae, TK1111 shared amino acids G 20, E 42, L 43, K 45, E 46, K 80, F 84, F 89, A 91, W 119, V 127, V 139, E 144, I 151, I 173, E 178, K 180, V 182, R 186, and K 188 (TK1111 numbering) with PGI from P. furiosus while these amino acids were replaced by N 20, K 42, M 43, E 45, G 46, N 80, M 84, Y 89, S 91, F 119, I 127, I 139, K 144, L 151, V 173, K 178, V 180, K 182, K 186, and R 188 in PGI from T. litoralis (Fig. 2). On the other hand E 39, I 57, E 116, and T 141 were identical in TK1111 and T. litoralis where as they were replaced by R 39, V 57, D 116, and I 141 in P. furiosus. These sequence replacements indicate the possibility of TK1111 being the prototype of PGIs in family Thermococcaceae. Expression of TK1111 gene in E. coli In order to analyze the function of TK1111, the encoding gene was cloned and expressed in E. coli. SDS-PAGE analysis of soluble and insoluble fractions of the cells containing pet-21a or pet-tk1111 revealed the presence of an additional band, corresponding to approximately 24 kda, in the soluble fraction of the cells containing pet-tk1111 which corresponded to the calculated molecular mass of TK1111 gene product. Production of TK1111 was more than 30% of the host proteins. Heat treatment at 85 C for 25 min resulted in the precipitation of the heat-labile host proteins leaving TK1111 in the supernatant. Purification of TK1111 was achieved to apparent homogeneity by ammonium sulphate precipitation and ion exchange chromatography (Fig. 3). Characterization of TK1111 Enzyme activity assay at various temperatures and ph showed that TK1111 displayed highest activity at 90 C (Fig. 4A) and ph 6.0 (Fig. 4B). To our knowledge TK1111 is the only PGI belonging to cupin-superfamily exhibiting highest enzyme activity in acidic ph. The extremely high temperature for highest enzyme activity and thermostability of TK1111 are in accordance with its function under the hyperthermophilic growth conditions of T. kodakarensis [7, 8]. Thermostability experiments demonstrated that TK1111 exhibits a half-life of 40 min at 90 C (data not shown) whereas PGI from P. furiosus has been reported highly thermostable with a half-life of 7.9 h at 95 C [15]. When we examined the effect of divalent metal ions on TK1111 enzyme activity we found that addition of Zn 2+ in the assay mixture remarkably enhanced the enzyme activity. There was more than 3-fold increase in enzyme activity in the presence of 200 μm ZnCl 2. Addition of Fig. 1. Phylogenetic analysis of TK1111 and its archaeal homologs. The tree was constructed by ClustalW provided by DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/). Segments responding to an evolutionary distance of 0.1 are shown. Accession numbers of the PGI sequences are: A. fulgidus, WP_ ; A. profundus, YP_ ; A. veneficus, YP_ ; F. placidus, YP_ ; M. fervens, YP_ ; M. jannaschii, AAB99624; M. vulcanius, YP_ ; P. aerophilum, WP_ ; P. arsenaticum, WP_ ; P. calidifontis, WP_ ; P. islandicum, YP_931140; P. abyssi, WP_ ; P. furiosus, NP_577925; P. horikoshii, NP_143784; T. barophilus, YP_ ; T. gammatolerans, YP_ ; T. litoralis, WP_ ; T. sibiricus, YP_ ; T. zilligii, WP_ ; TK1111, YP_

5 T.litoralis MKYKEPFGVKLDFETGIIENAKKSVRRLSDMKGYFIDEEAWKKMVEEGDPVVYEVYAIEQ 60 P.furiosus MYKEPFGVKVDFETGIIEGAKKSVRRLSDMEGYFVDERAWKELVEKEDPVVYEVYAVEQ 59 TK1111 MEYKRPIGLDIDLETGVIPGAKKLVRRLSDLKGYFLDEEAYNELL-KEDPVVYEVYAIEQ 59 ** * * * *** * *** ****** *** ** * ********* ** * ** * * * T.litoralis EEKEGDLNFATTVLYPGKVGNEFFMTKGHYHSKIDRAEVYFALKGKGGMLLQTPEGEARF 120 P.furiosus EEKEGDLNFATTVLYPGKVGKEFFFTKGHFHAKLDRAEVYVALKGKGGMLLQTPEGDAKW 119 TK1111 EEKEGDLNFATTVLYPGKVGKEFFFTKGHFHAKPDRAEIYYGIKGKGGMLLQTPEGEAEW 119 ******************** *** **** * * **** * ************* * Motif I * * T.litoralis IEMEPGTIVYVPPYWAHRTINTGDKPFIFLALYPADAGHDYGTIAEKGFSKIVVEENGKV 180 P.furiosus ISMEPGTVVYVPPYWAHRTVNIGDEPFIFLAIYPADAGHDYGTIAEKGFSKIVIEENGEV 179 TK1111 IPMGPGTVVYVPPYWAHRTVNTGNEPFIFLAIYPADAGHDYGSIKEKGFSKIVIEEDGEV 179 * * *** *********** * * ****** ********** * ******** ** * * Motif II T.litoralis VVKDNPKWRM 190 P.furiosus KVVDNPRWKK 189 TK1111 KVVDNPRWKE 189 * *** * Fig. 2. Multiple sequence alignment of TK1111 and other two characterized PGIs from family Thermococcaceae. Metal ion binding motif I and II, conserved in cupin-family PGIs, are shown in bold. Active site residues found in the cupin-family PGIs are shown by asterisks at the top. Amino acids involved in metal binding in P. furiosus PGI are shown with filled circles at the top. Amino acids identical in all the three sequences are shown by asterisks at the bottom. Fig. 3. Coomassie brilliant blue stained 14% SDS-PAGE demonstrating production and purification of recombinant TK1111. Lane M, protein marker (#SM0661, Thermo Scientific); lane 1, lysate of cells containing pet-21a plasmid; lane 2, lysate of cells containing pet-tk1111 plasmid; lane 3, purified TK1111. Mn 2+ and Fe 2+ also enhanced the enzyme activity but to a lesser extent. Enhancement of enzyme activity in the presence of Zn 2+ is in contrast with PGI from T. litoralis where Zn 2+ is reported to inhibit the enzyme activity [4]. The enzyme activity of PGI from P. furiosus, another member of the same family, was not affected in the presence of Zn 2+. However, the enzyme activity was remarkably enhanced in the presence of Mn 2+ [14]. The variable preference of the three PGIs from family Thermococcaceae towards metal ions may be attributed to the variability in the amino acid sequence in motif 1 and 2. It has been shown that the two histidine residues (H 88 and H 90, P. furiosus numbering) and a glutamate residue (E 97 ) in motif 1 together with the histidine residue (H 136 ) in motif 2 act as ligands for the binding of an active-site metal ion, such as Fe 2+, Mn 2+, or Zn 2+ [14, 16]. These residues are completely conserved in all the three characterized PGIs from family Thermococcaceae. However, residues after E 97 in motif 1 are quite different in all the three PGIs which may contribute to metal preference (Fig. 2). Presence of EDTA in the reaction mixture or measurement of enzyme activity after dialysis against 5 mm EDTA reduced the enzyme activity of TK1111. The enzyme activity was completely diminished when TK1111 was heated at 70 C in the presence of 10 mm EDTA. Complete restoration of the enzyme activity was observed when TK1111, after EDTA treatment and extensive dialysis, was incubated at room temperature in the presence of Zn 2+. Mn 2+ and Fe 2+ were also able to restore the activity but to a lesser extent (Fig. 5). Kinetic parameters TK1111 displayed a specific activity of 43 U/mg under optimal conditions, 90 C and ph 6.0, when fructose 442

6 Fig. 4. Effect of temperature and ph on TK1111 enzyme activity. A) Optimal temperature of the enzyme activity. Assays were performed at various temperatures ranging from 37 to 95 ºC under standard conditions. B) Optimal ph for TK1111 enzyme activity. Assays were conducted in various buffers at 50 ºC. Buffers used were: acetate buffer, open circles; phosphate buffer, closed circles; Tris-Cl buffer, open squares. basis of monomeric form of TK1111, was found to be 5 s -1 and k cat /K m value was 2.27 mm -1 s -1 at 37 C. k cat value at 90 C was 28 s -1. Kinetic data cannot be compared with other members of the family Thermococcaceae as analyses were performed at different temperatures. In summary, TK1111 belongs to the cupin-family of PGIs and is the only PGI from this family that exhibits highest activity in the acidic ph. Enzyme activity of TK1111 was remarkably enhanced in the presence of Zn 2+ which is in contrast to the other PGIs characterized from this family. Further characterization, particularly elucidation and stereochemistry of the reaction catalyzed by TK1111, is in progress. Conflict of Interest: There is no conflict of interest between the authors. References Fig. 5. Effect of metal ions on the restoration of TK1111 enzyme activity. TK1111 was heated at 70 ºC in the presence of 10 mm EDTA for 10 min and dialyzed extensively to remove the EDTA bound metal ions. Enzyme activity was measured after addition of 200 μm ZnCl 2 or MnCl 2 or FeCl 2. 6-phosphate was used as substrate. The enzyme activity was 6-times lower at 37 C which is quite similar with PGI from P. furiosus [3]. K m and V max values of TK1111 towards fructose 6-phosphate, determined from Lineweaver-Burke plot, were 2.2 mm and 12.5 mmol min -1 mg -1, respectively at 37 C. k cat, calculated on the [1] Hansen T, Oehlmann M, Scho nheit P. Novel type of glucose-6- phosphate isomerase in the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 2001; 183: [2] Hansen T, Wendorff D, Schonheit P. Bifunctional phosphoglucose/ phosphomannose isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum constitute a novel enzyme family within the phosphoglucose isomerase superfamily. J. Biol. Chem. 2004; 279: [3] Verhees CH, Huynen MA, Ward DE, Schiltz E, de Vos WM, van der Oost J. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin-superfamily. J. Biol. Chem. 2001; 276: [4] Jeong JJ, Fushinobu S, Ito S, Jeon BS, Shoun H, Wakagi T. Characterization of the cupin-type phosphoglucose isomerase from 443

7 the hyperthermophilic archaeon Thermococcus litoralis. FEBS Lett. 2003; 535: [5] Hansen T, Schlichting B, Felgendreher M, Scho nheit P. Cupintype phosphoglucose isomerases (Cupin-PGIs) constitute a novel metal-dependent PGI family representing a convergent line of PGI evolution. J. Bacteriol. 2005; 187: [6] Yeom SJ, Kim YS, Oh DK. Development of novel sugar isomerases by optimization of active sites in phosphosugar isomerases for monosaccharides. Appl Environ Microbiol. 2013; 79: [7] Morikawa M, Izawa Y, Rashid N, Hoaki T, Imanaka T. Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl. Environ. Microbiol. 1994; 60: [8] Atomi H, Fukui T, Kanai T, Morikawa M, Imanaka T. Description of Thermococcus kodakaraensis sp. nov., a well-studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1. Archaea 2004; 1: [9] Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S, Imanaka T. Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res. 2005; 15: [10] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J. Mol. Biol. 1990; 215: [11] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22: [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72: [13] Hansen T, Schlichting B, Grötzinger J, Swan MK, Davies C, Schönheit P. Mutagenesis of catalytically important residues of cupin-type phosphoglucose isomerase from Archaeoglobus fulgidus. FEBS J. 2005; 272: [14] Berrisford JM, Akerboom J, Turnbull AP, de Geus D, Sedelnikova SE, et al. Crystal structure of Pyrococcus furiosus phosphoglucose isomerase. Implications for substrate binding and catalysis. J. Biol. Chem. 2003; 278: [15] Yoon RY, Yeom SJ, Park CS, Oh DK. Substrate specificity of a glucose-6-phosphate isomerase from Pyrococcus furiosus for monosaccharides. Appl Microbiol Biotechnol. 2009; 83: [16] Anand R, Dorrestein PC, Kinsland C, Begley TP, Ealick SE. Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 Å resolution. Biochemistry 2002; 41:

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